We aim to understand the physical basis for the regulation of RNA polymerase II (Pol II) machinery and the crosstalk between the transcription and RNA processing pathways. The machinery is exceedingly large, consisting of Pol II at its heart and layers of trans-acting regulatory factors that modulate the Pol II activities. The advent of the crystal structure of Pol II core enzyme (513 kDa) has made it feasible to unravel the apparatus in more elaborative fashions. We have developed a structural paradigm in which functional Pol II complexes containing trans-acting regulators can be determined and mechanistic hypotheses emerging from the structures tested biochemically and genetically. Transcription is the first step in gene expression. Signals from many cellular processes such as nutrient sensing, catabolite repression, hormone stimulation, cell differentiation and responses to environmental stimuli ultimately reach the level of transcription to exert their effects. Aberration of transcriptional control is one of the mechanisms underlying tumorigenesis. The main characteristic of cancer cells is that they are highly proliferative due to their abilities to evade the programmed lifetime limit on normal cells. This means disruption of normal genetic programs, or more precisely, malfunction in the expression of genetic information stored in the DNA. As such, oncogene products are frequently found to be key players in the control networks of signal transduction, cell cycle progression and transcription regulation. The early elongation process of Pol II has been recognized as a major rate-limiting step in transcription regulation. The process is closely linked with cotranscriptional RNA processing, histone modification, and RNA export by mechanisms that are not yet clear. We propose to elucidate structures of Pol II early-elongation complexes to help understand the molecular underpinning of the central regulatory transition that occurs during the early phase of transcription. Specific aim are: (1) Determination and verification of the crystal structures of Spt5-Spt4-Pol II elongation complexes; (2) Defining the mechanism of cotranscriptional capping within the early elongation complex; and (3) Crystallization of Spt5/4-Pol IIO-CE, the complete early elongation/capping complex. Results will illuminate how interactions from trans-acting regulator (e.g., Spt5-Spt4) bound at the surface of Pol II control DNA/RNA transactions at the interior of the polymerase - whether there is a conformational relay system among the cis-acting Pol II subunits. The work will also determine the mechanistic parameters underlying cotranscriptional RNA 5' capping and assess the hypothesis of expanded Pol II spatial organization induced by the recruitment of processing factors. The proposed research will finally integrate the functional players of early elongation to allow for structural elucidation of the holo-complex that mediates the RNA processing-coupled transcription in the nucleus. PUBLIC HEALTH RELEVANCE: This project analyzes the structural and biochemical properties of the gene-reading protein machine - the RNA polymerase II transcription system. Focus is placed on understanding how trans-acting regulators control activities at the interior of the polymerase to modulate gene transcription.